Polypeptides and methods for the specific detection of antibodies in patients with a borrelia infection

ABSTRACT

The present disclosure relates to proteins derived from OspC from bacteria of the genus  Borrelia , in particular a protein which comprises a first OspC polypeptide, wherein the first OspC polypeptide is linked to a second OspC polypeptide via a disulphide bridge. The disclosure also relates to a method for the detection of antibodies against OspC and a method for the detection of a  Borrelia  infection, wherein a protein according to the disclosure is employed, and also to a diagnostic kit and a vaccine against  Borrelia.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119 of EuropeanPatent Application No. 08021660.9, filed Dec. 12, 2008, where thisEuropean patent application is incorporated herein by reference in itsentity.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 310159_(—)404_SEQUENCE_LISTING.txt. The textfile is 30 KB, was created on Dec. 11, 2009, and is being submittedelectronically via EFS-Web.

BACKGROUND

1. Technical Field

The present disclosure relates to polypeptides derived from OspC (“outersurface protein C”) from bacteria of the genus Borrelia, in particular aprotein which comprises a first OspC polypeptide, wherein the first OspCpolypeptide is linked to a second OspC polypeptide via a disulphidebridge. The disclosure also relates to a method for the detection ofantibodies against OspC and a method for the detection of a Borreliainfection, wherein a protein according to the disclosure is employed,and also to a diagnostic kit and a vaccine against Borrelia.

2. Description of the Related Art

Bacteria of the genus Borrelia are described as pathogens of severalhuman diseases, in particular as pathogens of Lyme borreliosis and ofrelapsing fever. An infection nowadays is usually detected viadetermination of specific antibodies against the bacteria in human oranimal body fluids. In this context, the presence of specific antibodiesagainst antigens which occur only in Borrelia indicates an infectionwith Borrelia. Early phases of infection, in particular fresh infectionsup to four weeks after initial contact, are characterized by thepresence of antibodies of the IgM class, in particular against theantigens OspC, p41 (flagellin) and P39 (BmpA), while late phases, inparticular infections which have run their course, have been cured orare chronically manifest, are accompanied by the presence of antibodiesof the IgG class, in particular against the antigens VIsE, p83/P100,p58, OspA, p41 (flagellin), P39, P18 and others (Wilske and Fingerle,2005).

An early diagnosis of the disease is particularly important becausetherapy with antibiotics is more successful and easier in the earlystages than in late stages, thus, e.g. in the early phase, an oraltherapy with antibiotics is still possible.

Antibodies against OspC of the IgM class are generally the mostimportant marker for an early phase of the disease. Native OspC is amembrane protein of the lipoprotein family 6 acylated with a fatty acid,which is anchored in the outer cell membrane (Norris et al., 1992;Hagman et al., 1998). During expression, in parallel with the cleavageof the signal sequence by signal peptidase II, the acyl residue isattached to the only conserved cysteine occurring in the OspC amino acidsequence (Wu and Tokunaga, 1986). It serves for anchoring in thelipid-containing cell membrane.

A conserved C-terminal peptide of ten amino acid residues, the terminalcarboxyl group of which must be freely accessible, is generally regardedas the most important epitope within OspC for reactions with humanantibodies of the IgM class (Mathiesen et al., 1998a; Mathiesen et al.,1998b), as is also described in the PCT patent specification WO 9742221.Epitopes in the poorly conserved central region of OspC are alsodescribed (Earnhart et al., 2005).

For detection of the antibodies, antigens purified from Borrelia areregularly used in immunobiochemical tests, such as ELISA, line blot orwestern blot. In the methods, individual or several antigens areconventionally bound to a solid phase and brought into contact with thebody fluid to be analysed, and the bound antibodies are detected by areporter molecule. Such kits are marketed e.g. by EUROIMMUN AG asEUROLINE-WB and Anti-Borrelia-plus-VIsE-ELISA. Similar methods aredescribed in the literature (Hansen et al., 1988; Cutler and Wright,1989; Fister et al., 1989).

Methods which are based on non-recombinant preparations can bereproduced only with a high outlay and are therefore cost-intensive,because essentially complex culture media which contain naturalconstituents which are not chemically defined, such asprotein-containing serum fractions from Mammalia and complex proteinmixture, e.g. proteolytically treated muscle extracts, yeast extracts orgelatine, are used for in vitro culturing of Borrelia. A commercial kiton this basis is obtainable e.g. from Sigma Aldrich (Complete BSK-H).Similar compositions are described in the literature with thedesignations MKP and BSK II (Ruzic-Sabljic and Strle, 2004).

Unfortunately, the constituents of these media which are not chemicallydefined are subject to marked variations in their compositions frombatch to batch, are at risk of contamination with viruses or Mycoplasmaand are cost-intensive to produce. Accordingly, Borrelia cultured invitro conventionally show marked variations in their growth rates andtheir gene expression patterns, depending on the particular culturemedia used or depending on individual components of the particularculture media used. Genes which are expressed in vivo, depending on theparticular host organism, are particularly affected by this (Pollack etal., 1993; Yang et al., 2001). This applies in particular to OspC. Theculturing temperature and the oxygen and carbon dioxide concentrationfurthermore also have a decisive influence on the nature and amount ofthe proteins expressed (Seshu et al., 2004; Hyde et al., 2007). As aresult, in particular the results from different laboratories whichculture Borrelia can be poorly compared with one another.

Methods which are based on individual antigens purified from Borreliacultured in vitro are furthermore susceptible to non-specific reactionscaused by the presence of further impurities which have not beenadequately depleted. In particular, in methods which only generate onesignal, such as, for example, ELISA or line blot, falsely positiveresults are thus regularly obtained. In contrast, western blots, whichbypass this problem by local resolution of the signals, have thedisadvantage that they generate a considerable additional effort due tothe required electrophoresis and the transfer to a membrane.

In further methods, antigens are used which are prepared by recombinanttechniques, e.g. by heterologous expression of antigens or antigenfragments in E. coli, as described e.g. in the European patentspecification EP 0506868. Commercially obtainable variants are e.g.EUROLINE-WB and Anti-Borrelia-plus-VIsE-ELISA (IgG) and the recomlineBorrelia IgM (Mikrogen). Similar methods are described in the literature(Hauser et al., 1998; Lawrenz et al., 1999; Wilske and Fingerle, 2005).In general, recombinant antigens have the advantage that they can bepurified in a more defined manner and with less effort than nativeantigens, e.g. by fusion with purification polypeptides, e.g. thepolyhistidine-tag (=His-tag).

Heterologous expression of OspC including its intrinsic signal sequencein E. coli and subsequent acylation is possible, but leads to a lowexpression efficiency (Fuchs et al., 1992). For this reason, in generaldeletion constructs of OspC with increased expression efficiency inwhich at least the signal sequence is missing are employed. In thiscontext, in the prior art, at least the first 19 amino acids of OspC,including the cysteine which occurs in the OspC amino acid sequence, aredeleted (Fuchs et al., 1992; Wilske et al., 1993; Wilske et al., 1995;Eicken et al., 2001; Kumaran et al., 2001), and this is in some casessupplemented by N-terminal fusion with heterologous polypeptides, e.g.the His-tag.

Such N-terminally shortened variants have also been used forclarification of the spatial structure of OspC (Kumaran et al., 2001;Eicken et al., 2001). For this, the first 31 (Eicken et al., 2001) or 37(Kumaran et al., 2001) amino acid residues of OspC were deleted in therecombinant OspC variants in order to achieve in particular a suitableamount and purity of the OspC. The two exemplary spatial structuresindicate that recombinant OspC tends to dimerize. This dimerizationtakes place on the basis of ionic interactions and hydrogen bridgebonds, but not by a covalent linking.

There are also attempts to prepare acylated variants of OspCrecombinantly by upstream insertion of a heterologous signal sequence,as described e.g. in the European patent specification EP 1741718. Byattaching the fatty acid, such OspC variants are intended to have ahigher in vivo immunogenicity than the N-terminally deleted or modifiedOspC variants described until then.

The recombinant variants of OspC used hitherto for diagnostics ingeneral have the disadvantage that they differ from the native OspCpurified from Borrelia cultured in vitro, because they are not or aredifferently modified post-translationally, and the presence ofconformational epitopes or the accessibility of epitopes in general isnot comparable or not guaranteed. The manifestation of thesedisadvantages is the lower specific reactivity of recombinant OspCvariants used to date compared with non-recombinant preparations, whichis regularly described in the literature. It is reflected in high ratesof false positive results in such diagnostic methods.

The development of recombinant OspC variants which, with an easier andmore defined preparation compared with the native antigens, can beemployed in diagnostic methods and at the same time do not lead to thedisadvantages described for the recombinant OspC variants to date istherefore necessary in order to facilitate the preparation of such invitro diagnostic agents or the implementation of such diagnostic methodsand to standardize the results of different laboratories.

BRIEF SUMMARY

In one aspect, the present disclosure provides a protein that comprisesa first OspC polypeptide and a second OspC polypeptide, wherein thefirst OspC polypeptide is linked to the second OspC polypeptide via adisulphide bridge.

In another aspect, the present disclosure provides a method fordetecting antibodies in a biological sample against the above-describedprotein, comprising: contacting the biological sample with the protein,and detecting binding of antibodies to the protein.

In another aspect, the present disclosure provides a method fordiagnosing a Borrelia infection, comprising contacting a biologicalsample of a patient with the above-described protein, and determiningthe presence or absence of binding of antibodies that may be in thesample to the protein, wherein the presence of the binding of antibodiesto the protein indicates a Borrelia infection.

In another aspect, the present disclosure provides a kit for diagnosinga Borrelia infection, comprising the above-described protein.

In another aspect, the present disclosure provides a method forvaccinating against a Borrelia infection, comprising administering theabove-described protein to a patient.

In another aspect, the present disclosure provides a vaccine against aBorrelia infection, comprising the above-described protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of mature OspC from B. afzeliiVS461=OspC_(VS461) (SEQ ID NO:3), from B. garinii 20047=OspC₂₀₀₄₇ (SEQID NO:6) and from B. burgdorferi B31=OspC_(B31) (SEQ ID NO:9),determined with CLUSTAL 2.0.10 with standard settings. The table showsthe degree of amino acid identity.

FIG. 2 shows by way of example a western blot with the non-reducedvariants His-m-OspC_(VS461) and His-m-Cys-OspC_(VS461) after stainingwith Ponceau S (track 1-3) and after incubation with a serum of apatient with a borreliosis in the early stage (1:200 dilution) anddetection of the bound IgM antibodies (track 4 & 5). Track 1: sizemarker Mark12 (Invitrogen), track 2 & 4: 15 μg of His-m-OspC_(VS461),track 3 & 5: 25 μg of His-m-Cys-OspC_(VS461).

DETAILED DESCRIPTION

It has now been found, surprisingly, that the problems described can besolved by the disclosure, in particular the subject matter of theclaims.

The disclosure provides a protein comprising a first OspC polypeptide,wherein the first OspC polypeptide is covalently linked to a second OspCpolypeptide. It has proved advantageous in the context of the disclosureif the first OspC polypeptide is linked to a second OspC polypeptide viaa disulphide bridge.

An OspC polypeptide in the context of the disclosure, in particular thefirst and/or second OspC polypeptide, can have an amino acid identity ofat least 70% with SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9 and isrecognized (in this homologous region) specifically by antibodiesagainst OspC from Borrelia. Preferably, the amino acid identity is atleast 80%, at least 90%, at least 95% or at least 99% to one or more ofthe sequences according to SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9.These sequences correspond to the native OspC sequences from the threetype strains of the Borrelia genospecies Borrelia afzelii (VS461),Borrelia garinii (20047) and Borrelia burgdorferi (B31). The amino acididentity between these strains is 70% and 76% (see FIG. 1). The aminoacid identity is preferably determined with the program CLUSTAL 2.0.10with standard settings. In the context of the disclosure, in particular,an OspC sequence from another Borrelia strain, e.g. PKo, or a variantthereof can be employed, the specific binding by antibodies against OspCfrom Borrelia being decisive.

In the context of the disclosure, a specific recognition by antibodiesagainst OspC from Borrelia means that under conditions suitable forthis, which are known to the person skilled in the art and described,for example, in the examples, antibodies against OspC from Borrelia bindto the polypeptide or protein, but not to another unrelated protein.Serum from patients with a confirmed early phase of a Borrelia infectioncan be employed, for example, as antibodies against OspC from Borrelia,as can antibodies which have been produced e.g. by immunization of miceor rabbits with native purified OspC (e.g. EUROIMMUN), in particularpolyclonal antibodies.

Borrelia includes all bacteria of the genus Borrelia, and B.burgdorferi, B. garinii and B. afzelii are mentioned by way of example.

An OspC polypeptide in the context of the disclosure, in particular thefirst and/or second OspC polypeptide, may comprise at least one epitopewhich is specifically recognized by antibodies against OspC fromBorrelia, the epitope comprising at least a partial sequence of 10contiguous amino acids from SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9, inparticular the 10 C-terminal amino acids of these sequences. Preferably,the partial sequence comprises the epitope described as immunodominantin Mathiesen et al., 1998a; Mathiesen et al., 1998b. In particular, theOspC polypeptide(s) comprise(s) one of the amino acid sequencesaccording to SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8 in the C-terminallocation. It is preferable for the OspC polypeptide(s) to end at theC-terminus with the sequence of the native OspC, that is to say nofurther amino acids which could impede the recognition of the C-terminalepitope are contained here.

Alternatively or in addition, one or more further epitopes, e.g. asdescribed by Earnhart et al., 2005, can be present in the OspCpolypeptide.

Preferably, the epitope is or the epitopes are embedded in furthersequences from OspC corresponding to a wild type sequence from OspC fromBorrelia. Preferably, the partial sequence of contiguous amino acidsfrom SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9 has a length of at least15, at least 20, at least 25, at least 30, at least 50, at least 100, atleast 150 or at least 190 amino acids. Preferably, these are theC-terminal amino acids from SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9,from homologues from other Borrelia strains or homologues with asequence identity of at least 70%, at least 80%, at least 90%, at least95% or at least 99% with SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9.

The OspC polypeptides according to the disclosure contain no signalsequences for an acylation. Polypeptides expressed in the prior art (EP1741718) which, like the wild type polypeptide, were to be acylated onthe furthest N-terminally located cysteine, were expressed with aheterologous signal sequence.

In contrast, the polypeptide according to the disclosure containsneither a homologous signal sequence nor a heterologous signal sequence.Signal sequences which lead to an acylation conventionally comprise aBraun's signal sequence which is recognized by signal peptidase II. Suchsignal sequences can comprise e.g. the sequence L-I-A-C (as OspA orOspB) or L-X-Y-C (wherein X and Y are small neutral amino acids) (Fuchset al., 1992). Preferably, the polypeptide according to the disclosurealso was not expressed with an acylation sequence, this then being splitoff. The OspC polypeptide according to the disclosure is therefore alsonot acylated. In one embodiment of the disclosure, the cysteine on whichthe acylation would take place in the wild type OspC is the cysteine onwhich the disulphide bridge to a further OspC polypeptide is formed.

Alternatively or in addition, a disulphide bridge can also be formed onanother cysteine. Preferably, the disulphide bridge is formed by bondingof a cysteine which is not more than about 100, preferably not more thanabout 50 or not more than about 30 amino acid positions from theN-terminus of the OspC polypeptide. In particular, the disulphide bridgecan be formed on a cysteine which forms the N-terminus of thepolypeptide or is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acidsaway from the N-terminus.

In order to facilitate the purification of the protein according to thedisclosure, in one embodiment, on the N-terminus, in particularN-terminally from the cysteine on which the disulphide bridge is formed,the OspC polypeptide/the OspC polypeptides contain/contains a His-tag,which can have e.g. the sequence corresponding to the first 12 aminoacids of SEQ ID NO:1. Instead of 8, e.g., 6 or 10 histidines can also beemployed. Other amino acid sequences known in the prior art whichfacilitate purification or detection of the protein, e.g. a Flag-tag,can also be incorporated here. Such sequences should not impede therecognition by antibodies against OspC from Borrelia. Under thisprerequisite, bonding or incorporation of a reporter molecule is alsopossible at another location in the molecule. The reporter molecule canbe chosen, e.g., from a group comprising His-tag, Flag-tag, afluorescence label, biotin and streptavidin.

In one embodiment, the first OspC polypeptide N-terminally comprises aHis-tag, which is adjacent to a cysteine which is bonded to the secondOspC polypeptide in the protein according to the disclosure via adisulphide bridge, and following this in the polypeptide chain of thefirst OspC polypeptide, the further sequence of an OspC polypeptide froma Borrelia strain, e.g. B. burgdorferi, B. afzelii or B. garinii, thissequence forming the C-terminus of the polypeptide. In particular, theOspC polypeptide can consist of these sequences. The second (andoptionally further) OspC polypeptide preferably comprises the samesequence constituents (His-tag-Cys-OspC sequence) or consists of these.

In a preferred embodiment, the first and/or the second OspC polypeptidecontains a sequence according to SEQ ID NO:2, SEQ ID NO:5 or SEQ IDNO:8.

In the context of the present disclosure, “a” in general is not used asa numerical word, but an indeterminate number can be described by this.Unless described otherwise, it can thus also represent two or more. Inparticular, in the context of the disclosure an OspC polypeptide isunderstood as meaning the first and/or the second OspC polypeptide of aprotein according to the disclosure. This does not exclude the presenceof further OspC polypeptides, which preferably have the same structureas the first OspC polypeptides.

Preferably, the proteins according to the disclosure are dimers of twoOspC polypeptides. Multimerization with further OspC polypeptides orother molecules (e.g. via additional disulphide bridges) is also inprinciple possible, as long as this does not impede or restrict therecognition by antibodies against OspC from Borrelia. It has beendemonstrated in the context of the disclosure that by means of thedisulphide bridge, a homodimer of two identical OspC polypeptides or aheterodimer of different OspC polypeptides can be formed. With a proteinaccording to the disclosure, it is therefore possible e.g. to conductthe detection of antibodies against several Borrelia strains, or toprepare a vaccine against several Borrelia strains.

With respect to the present disclosure, protein is in general used formultimers of OspC polypeptides and polypeptide for individual OspCpolypeptide chains, but both terms, as can be seen by the person skilledin the art from the context in each case, can also relate to proteins,polypeptides or peptides according to general scientific usage, nodifferentiation being made between different sizes.

In the context of the disclosure, it has been demonstrated that thedisulphide bridge bond directly between the OspC polypeptidessignificantly improves the recognition by antibodies against OspC fromBorrelia. Associated with this, the recognition of the protein byantibodies against OspC from Borrelia is reduced by bringing it intocontact with at least one reagent which destroys the disulphide bridge.In particular, such a reagent is chosen from a group comprisingthiol-containing reagents or a combination of a thiol-containing reagentand an alkyl halide. Such reagents include e.g. reducing agents, such asdithiothreitol or mercaptoethanol, optionally in combination with analkylating agent, such as iodoacetamide. In particular, a reducing agentcan first be employed, and then an alkyl halide.

A protein according to the disclosure can be produced e.g. in bacterialcells (e.g. E. coli, e.g. E. coli RosettaBlue(DE3)pLacl (Stratagene)),insect cells (e.g. SF6 cells), yeast cells (e.g. S. cerevisiae) or cellsof vertebrates (e.g. CHO cells). The disclosure includes a method forthe production of the protein according to the disclosure wherein thisis recombinantly expressed and purified. Purification is possible e.g.via affinity chromatography, it being possible to use, for example,antibodies against OspC from Borrelia or—if e.g. a His-tag is used—anickel affinity chromatography. Preferably, non-reducing conditions areused for the expression and/or purification. Preferably, a pH of 7-9, apH of 7.5-8.5, in particular pH 8 is used during the purification. In anaffinity chromatography e.g. the mixture to be purified can be appliedand washed with TNI10 buffer, and the protein can be eluted with TNI150buffer. Other production and purification methods which do not impedethe formation of the disulphide bridge are known in the prior art.

In one aspect, the present disclosure relates to a method for thedetection of antibodies against a protein according to the disclosure,in which a biological sample is brought into contact with the proteinaccording to the disclosure, and binding of antibodies, which maypossibly be present in the sample, to the protein is detected.

In particular, a method for the diagnosis of a Borrelia infection isdisclosed, in which a biological sample from a patient is brought intocontact with a protein according to the disclosure, and binding ofantibodies which may possibly be present in the sample to the protein isdetected, wherein detection of the binding of antibodies indicates aBorrelia infection.

The disclosure also provides the use of a protein according to thedisclosure for the detection of antibodies against OspC of Borrelia andfor the diagnosis of a Borrelia infection. The antibodies detected arepreferably antibodies of the IgM type, by which means a Borreliainfection in an early phase is detected.

The binding of the antibodies can be detected e.g. with animmunofluorescence test, ELISA, luminescence test, western blot, lineblot or dot blot. The biological sample employed can be e.g. a bodyfluid of a patient, in particular blood, serum, plasma, saliva, urine orcerebrospinal fluid. Preferably, the patient is a human or animalpatient who is to be tested, for example, for an infection withBorrelia.

The disclosure also provides a kit for the diagnosis of Borreliainfections comprising a protein according to the disclosure. Such a kitcan furthermore comprise antibodies against OspC from Borrelia and/orbuffers and/or reagents suitable for the detection, such as e.g. anantibody which recognizes human IgM, optionally marked with a reagentsuitable for the detection (e.g. a fluorescent dye, such as FITC or PE,an enzyme, such as alkaline phosphatase or horseradish peroxidase, orbiotin). It can be e.g. a kit for an immunofluorescence test, ELISA,luminescence test, western blot, line blot or dot blot.

The disclosure also provides the use of a protein according to thedisclosure for the preparation of a vaccine against a Borreliainfection, and a vaccine against a Borrelia infection comprising aprotein according to the disclosure. Such a vaccine may comprisesuitable auxiliary substances, buffers and/or adjuvants. It can be acombination vaccine which, for example, can be directed against two,three or more Borrelia strains. This can be achieved by the use ofheteromeric OspC proteins, but also e.g. by mixing various homodimers.Such a vaccine can be formulated e.g. for subcutaneous, intramuscular,intravenous or oral administration. Multiple administration, e.g. twice,three times or four times (for example on day 1, 14, 28, 42), may beappropriate in order to improve the formation of antibodies.

The present disclosure for the first time makes it possible to determineOspC-specific antibodies with the aid of recombinant OspC variants whichgive results which are diagnostically equivalent to the use ofnon-recombinant OspC variants. At the same time, the use of the novelrecombinant OspC variants with disulphide bridges makes a considerablereduction in the effort for the production of the antigens possible,compared with the native OspC variants which were hitherto regarded asdiagnostically the most competent.

It has been found that the OspC variants according to SEQ ID NO:1, SEQID NO:2 (derived from mature OspC according to SEQ ID NO:3 from B.afzelii VS461=OspC_(VS461), Genbank Accession No. D49379), SEQ ID NO:4,SEQ ID NO:5 (derived from OspC according to SEQ ID NO:6 from B. garinii20047=OspC₂₀₀₄₇, Genbank Accession No. L42900), SEQ ID NO:7 and SEQ IDNO:8 (derived from OspC according to SEQ ID NO:9 from B. burgdorferiB31=OspC_(B31), Genbank Accession No. U01894) can be expressed in largeamounts in E. coli and can be prepared with a high purity. No differencewas found in this context in the effort for the genetic engineeringwork, expression rate, effort for the preparation or purity between thevariants according to SEQ ID NO:1 and SEQ ID NO:2 (derived fromOspC_(VS461)), SEQ ID NO:4 and SEQ ID NO:5 (derived from OspC₂₀₀₄₇) andSEQ ID NO:7 and SEQ ID NO:8 (derived from OspC_(B31)), which in eachcase differ by only one cysteine.

It has moreover been demonstrated by way of example that the diagnosticaccuracy of a method for the determination of antibodies of the IgMclass using the variant according to SEQ ID NO:2 derived fromOspC_(VS461) surprisingly is equivalent to an established method usingnon-recombinant OspC of the strain VS461. It has furthermore beendemonstrated that the diagnostic accuracy of these two methods isgreater than in methods which use the variant according to SEQ ID NO:1derived from OspC_(VS461) or recombinant OspC variants deleted inaccordance with the scientific literature.

It has furthermore been demonstrated by way of example that the variantaccording to SEQ ID NO:2 derived from OspC_(VS461) is present in a formdimerized via a disulphide bridge after biochemical purification, andthat it surprisingly has a higher specific reactivity than its monomer,its monomer acetylated on the cysteine and the analogous monomeraccording to SEQ ID NO:1.

It has furthermore been demonstrated by way of example that by mixingthe variants according to SEQ ID NO:2 (derived from OspC_(VS461) and SEQID NO:5 (derived from OspC₂₀₀₄₇), followed by reduction thereof andsubsequent oxidation, heterodimers with disulphide bridges can beprepared, the usability of which is in principle equivalent to thehomodimers with disulphide bridges, but which have the advantage ofbeing able to detect several different species-specific antibodies, i.e.antibodies directed exclusively against OspC from B. afzelii strains orexclusively against OspC from B. garinii strains. Means and methods forseparating heterodimers from homodimers, e.g. by isoelectric focussing,are accessible to the person skilled in the art. Such a separation ishowever not necessary, since mixtures of homodimers and heterodimers canalso be used.

It has furthermore been demonstrated by way of example that micesurprisingly have a higher titre of antibodies against Borrelia afterimmunization with the OspC variant according to SEQ ID NO:2 (derivedfrom OspC_(VS461)) dimerized via disulphide bridges than afterimmunization with the same amount of the monomeric OspC variantaccording to SEQ ID NO:1. A higher effectiveness of vaccines based onthe polypeptides according to the disclosure can be concluded from this.The results can be applied to the other variants, to other animals andto humans.

The following examples present the subject matter of the disclosure byway of example, but are not intended to limit the disclosure.Modifications are obvious to the person skilled in the art. All thepublications cited are included explicitly by the reference.

EXAMPLES Example 1 Cloning of Vectors for Expression of Ospc in E. Coli

OspC is coded on plasmid B in Borrelia (nomenclature on the basis of thecomplete B. burgdorferi genome, reading frame BB_B19). Total DNA from B.afzelii strain VS461, B. garinii strain 20047 and B. burgdorferi strainB31 is used as the template for PCR amplification of gene regionsaccording to SEQ ID NOS:10 to 15 coding for six OspC variants. Theprimer combinations according to SEQ ID NO:16 and SEQ ID NO:17(His-m-OspC_(VS461)), SEQ ID NO:18 and SEQ ID NO:17(His-Cys-m-OspC_(VS461)), SEQ ID NO:19 and SEQ ID NO:20(His-m-OspC₂₀₀₄₇), SEQ ID NO:21 and SEQ ID NO:20 (His-Cys-m-OspC₂₀₀₄₇),SEQ ID NO:22 and SEQ ID NO:17 (His-m-OspC_(B31)) and SEQ ID NO:23 andSEQ ID NO:17 (His-Cys-m-OspC₃₁) are employed for amplification of theindividual variants. By the PCR, the necessary restriction cleavagesites are integrated into the amplificates at the 5′ and 3′ end of thecoding strand, respectively.

The PCR is carried out on a 50 μl scale using the “High Fidelity PCREnzyme Mix” (Fermentas) according to the manufacturer's instructions. 30reaction cycles composed of steps 1 to 3 are carried out:

Step 1 30 seconds 94° C. Step 2 45 seconds 56° C. Step 3 30 seconds 72°C.

The PCR fragments are then purified using the NucleoSpin® Extract IIpurification system (MACHEREY-NAGEL GmbH & Co. KG) in accordance withthe manufacturer's instructions and taken up in each case in 50 μl of 5mM Tris-HCl pH 8.5. The fragments are then cleaved at the ends byrestriction endonucleases. The restriction endonucleases are obtainedfrom Fermentas and employed in accordance with the manufacturer'sinstructions.

The amplificates are treated with restriction endonucleases as follows:

His-m-OspC_(VS461): Ncol and Xhol,

His-Cys-m-OspC_(VS461): Esp3I and Xhol,

His-m-OspC₂₀₀₄₇: Ncol and Xhol,

His-Cys-m-OspC₂₀₀₄₇: Esp3I and Xhol,

His-m-OspC_(B31): Ncol and Xhol,

His-Cys-m-OspC_(B31): Pagl and Xhol.

The PCR fragments are then purified using the NucleoSpin® Extract IIpurification system in accordance with the manufacturer's instructionsand taken up in 50 μl of 5 mM Tris-HCl pH 8.5, respectively.

The enzymatically cleaved and purified PCR fragments are then integratedinto Ncol/Xhol-cleaved plasmid according to SEQ ID NO:24 by ligation.The Rapid DNA Ligation Kit from Fermentas is employed for the ligationin accordance with the manufacturer's instructions. The ligation batchis transformed into E. coli RosettaBlue(DE3)pLacl (Stratagene).

Positive clones are selected on the basis of kanamycin/chloramphenicol[50/34 μg/ml] resistance. The plasmids contained in these clones areisolated from them and checked by restriction analysis and DNAsequencing. Correct plasmids are chosen for carrying out the expression,inoculated into 20 ml of LB medium containing antibiotics and incubatedat 37° C. until an OD600 (d=1 cm) of 0.6 is reached. The proteinexpression is induced by addition of IPTG (final concentration: 1 mM)and incubation is then carried out for a further 3 hours at 37° C. Afterthis time, the cells are sedimented by centrifugation at 2,200×g for 10minutes, resuspended in 20 ml of PBS, centrifuged again and finallyresuspended in 1 ml of PBS.

The cells are broken down by addition of one third volume of4×NuPAGE-LDS sample buffer (Invitrogen), containing 141 mM Tris-HCl pH8.5, 2%) (w/v) lithium dodecyl sulphate, 10% (w/v) glycerol, 0.51 mMEDTA, 0.22 mM SERVA Blue G250, followed by incubation for 10 minutes at70° C. Chromosomal DNA is then fragmented by ultrasound treatment(Branson Sonifier, level 7, MicroTip).

After separation by SDS-PAGE, the cell lysate is transferred to anitrocellulose membrane. Non-saturated positions of the membrane arethen blocked by incubation with “universal buffer” (EUROIMMUN) with 3%(w/v) milk powder for 15 minutes. Incubation is then carried out for 1hour with a monoclonal antibody from the mouse (Merck Biosciences GmbH)directed against the His-tag and diluted 1:2,000 in “universal buffer”with 3% (w/v) milk powder. Washing is then carried out with “universalbuffer” three times for 5 minutes each time. In a second incubationstep, the antibodies bound to the proteins in the positive case reactwith a conjugate solution which is diluted 1:2,000 in “universal buffer”and contains as the conjugate an anti-mouse IgG antibody labelled withalkaline phosphatase (Sigma). Washing is then carried out as after theanti-His-tag incubation. In a third incubation step, the boundantibodies are then detected with an NBT/BCIP “substrate solution”(4-nitroblue tetrazolium chloride/chlorobromoindolyl phosphate,EUROIMMUN).

Results:

In the western blot after reducing SDS-PAGE, the expression of therecombinant constructs can be detected by the presence ofHis-tag-reactive proteins, the sizes of which are in good agreement withthe masses of 23-25 kDa predicted on the basis of the amino acidsequences. Cells which contain the unchanged plasmid vector contain nocorresponding protein.

Example 2 Purification of Recombinant Ospc Variants by AffinityChromatography

The affinity column is a NiNTA spin column (Qiagen) handled inaccordance with the manufacturer's recommendations.

The cells harvested according to Example 1 are centrifuged again asdescribed, resuspended in 1 ml of “TNI10 buffer” (5 mM Tris-HCl pH 8.0;300 mM NaCl; 10 mM imidazole) and broken down by three ultrasoundtreatments of one minute (Branson Sonifier, level 7, MicroTip). 0.5 mlof the supernatant is applied to a NiNTA spin column equilibrated with“TNI10 buffer”.

Non- or weakly bound proteins are removed from the column by washingseveral times with 0.5 ml of “TNI10 buffer”. Elution is carried out with0.2 ml of “TNI150 buffer” (5 mM Tris-HCl pH 8.0; 300 mM NaCl; 150 mMimidazole). The elution fraction is analysed by western blot as inExample 1.

Results:

The eluates essentially contain proteins which, after staining of thewestern blot membrane with Ponceau S staining solution, in each casecorrespond to bands corresponding to a size of about 23-25 kDa. Afterwestern blot and detection of the His-tag, a bond corresponding to thesame size can be seen. This result indicates that after thechromatography, the protein expressed is present in a form essentiallyfree from E. coli constituents.

Example 3 Line Blots for Determination of Anti-ospc Antibodies with theAid of the Recombinant Ospc Variants

Nitrocellulose membranes are coated in lines with a purified recombinantOspC according to Example 2 diluted in 10% (w/v) glycerol(concentration: 20-100 μg/ml). The membranes are then dried overnight,blocked for 1 hour with “universal buffer” (EUROIMMUN), fixed on a solidphase, cut into strips about 2 mm wide with a scalpel at a 90° angle tothe OspC lines, and then stored at 4° C. in aluminium bags withdesiccant bags until used. The strips are incubated as described in theworking instructions for EUROLINE-WB strips (EUROIMMUN), which areincubated as a comparison. Incubation of recomLine Borrelia strips(Microgen) in accordance with the manufacturer's instructions serves asa further comparison.

For determination of the sensitivity and specificity, 25 serum samplesfrom patients with confirmed early phase of a Borrelia infection and 50serum samples from pregnant women (which frequently contain non-specificantibodies) without signs of a Borrelia infection and 25 serum samplepairs from healthy persons taken at an interval of 14 days are analysedwith respect to the presence of OspC-specific antibodies ofimmunoglobulin class M (IgM). Positive reactions in the healthy personsare checked by analysis of the IgG seroconversion (comparison of day0/day 14) with several test systems for detection of antibodies of theIgG class against Borrelia, e.g. EUROLINE-WB IgG and anti-Borrelia ELISAIgG (EUROIMMUN). In no case of positive reaction, the follow-up samplegave indications of seroconversion.

TABLE 1 His-Cys-m- His-m- recomline EUROLINE- OspC_(VS461) OspC_(VS461)Borrelia IgM WB IgM SEQ ID NO: 2 SEQ ID NO: 1 Borrelia afzelii nAnti-OspC IgM Early phase [n] 20 17 17 17 19 Sensitivity [%] 20 85 85 8595 Healthy pregnant women [n] 50 2 2 6 8 Healthy blood donors [n] 25 4 47 9 Specificity [%] 75 92 92 82.7 77.3 Diagnostic accuracy [%] 100 90.590.5 83.2 81.1

Results:

The results are summarized by way of example for OspC from B. afzelii inTable 1.

A large proportion of the sera of patients in an early phase of Borreliainfection contain IgM antibodies against OspC. In the study, the westernblot with the native OspC originating from B. afzelii and the line blotwith His-Cys-m-OspC_(VS461) have virtually equivalent sensitivities andspecificities and at the same time the highest diagnostic accuracies.The line blot with the variant His-m-OspC_(VS461) and the recombinantOspC from B. afzelii on the recomBlot Borrelia IgM, in contrast, aredistinguished above all by non-specific reactions in the healthypersons.

Example 4 Analysis of the Recombinant Ospc Variants

In accordance with Example 2, the various purified OspC variants areseparated by SDS-PAGE. In this context, the separation is carried outboth under non-reducing conditions, under reducing conditions in thepresence of dithiothreitol (DTT) and under alkylating conditions in thepresence of iodoacetamide after DTT reduction. Thereafter, a westernblot is carried out in accordance with Example 1. In parallel with themonoclonal antibodies against the His-tag described in Example 1,anti-OspC IgM positive sera are also incubated in a 1:200 dilution inaccordance with Example 3. In the case of human sera, a conjugate ofanti-human IgM and alkaline phosphatase (EUROIMMUN) diluted 1:10 in“universal buffer” is used in the second incubation step.

Results:

The separation under reducing and alkylating conditions, both afterPonceau S staining and after detection of the His-tag in accordance withExample 1, in each case leads to bands corresponding to a size of about23-25 kDa. This also applies to the variants His-m-OspC_(VS461) (SEQ IDNO:1), His-m-OspC₂₀₀₄₇ (SEQ ID NO:4) and His-m-OspC_(B31) (SEQ ID NO:7)under non-reducing conditions. In contrast, in the case of the variantsHis-Cys-m-OspC_(VS461) (SEQ ID NO:2), His-Cys-m-OspC₂₀₀₄₇ (SEQ ID NO:5)and His-Cys-m-OspC_(B31) (SEQ ID NO:8), in each case two bands arepresent at 23-25 and 45-50 kDa, which are each stained approximatelywith the same intensity. These results indicate that the variantsHis-Cys-m-OsPC_(VS461), His-Cys-m-OspC₂₀₀₄₇ and His-Cys-m-OspC_(B31) areeach present as dimmers, in which in each case two monomers are linkedto one another via disulphide bridges (see FIG. 2).

After incubation with human sera, the distribution of the bands in theparticular variants is identical. It is striking, however, that thebands at 45-50 kDa are in each case stained considerably moreintensively than the bands at 23-25 kDa. The different colourintensities after direct staining by Ponceau S and immunologicalstaining after incubation with human sera indicate that the dimers ineach case have higher specific reactivities than the monomers (see FIG.2).

Example 5 Preparation of Heterodimers

Aliquots of two of the OspC dimer variants purified in accordance withExample 2, e.g. His-m-Cys-OspC_(VS461) and His-m-Cys-OspC₂₀₀₄₇ are mixedwith one another in equal portions. An aliquot of the mixture is storedfor analysis. The remainder is reduced by addition of DTT and thesuccess of the reduction is checked by non-reducing SDS-PAGE and westernblot in accordance with Example 4. The mixture is then freed from DTT byexhaustive dialysis against 20 mM Tris-HCl pH 8.5 and compressed air ispassed through for 24 hours through a pipette tip in order to create anoxidative medium. The mixture is finally analysed by non-reducingisoelectric focussing between pH 6 and 9 with the ZoomRunner system(Invitrogen) in accordance with the manufacturer's instructions,subsequent reducing SDS-PAGE and western blot. The two individual OspCdimers and the mixture before addition of DTT are analysed forcomparison.

Results:

His-m-Cys-OspC_(VS461) has a spot corresponding to approx. pH 7.5 and 24kDa, His-m-OspC₂₀₀₄₇ on the other hand corresponding to approx. pH 6.5and 24 kDa. The mixture of His-m-OspC_(VS461) and His-m-OspC₂₀₀₄₇ beforereduction has two spots corresponding to approx. pH 7.5/24 kDa and pH6.5/24 kDa. In contrast, the reduced and subsequently air-oxidizedmixture has three spots corresponding to approx. pH 7.5/24 kDa, pH6.8/24 kDa and pH 6.5/24 kDa. The results indicate that heterodimers areat least partly present.

Example 6 Immunogenicity of Ospc Dimers with Disulphide Bridges

The proteins His-m-OspC_(VS461) and His-Cys-m-OspC_(VS461) purified inaccordance with Example 2 were injected subcutaneously without anadjuvant into 10 female C3H/He mice, respectively, aged from four to sixweeks for the first time and then at an interval of 14, 28 and 42 daysin portions of in each case 100 μg per dose. Further 10 mice areinjected with 0.9% (w/v) saline solution as a control, respectively.Blood is taken from all animals on the day of the first injection andthen after 24, 38, 52 and 66 days. OspC-specific antibodies aredetermined in a 1:1,000 dilution of the particular serum with the aid ofan anti-Borrelia IgM ELISA (EUROIMMUN), in which the conjugate fordetection of bound human antibodies is exchanged for a conjugate,diluted 1:2,000 in conjugate dilution buffer (EUROIMMUN), for detectionof bound murine antibodies of the IgG class (Dianova). The mean (MW) andstandard deviation (a) of the results of in each case 10 animals treatedin the same way are determined.

To check the specificity of the immune response, the 30 serum samplesfrom the day of the last removal of blood are incubated on EUROLINE-WBstrips (EUROIMMUN) in a 1:500 dilution and the bound murine antibodiesare detected in accordance with experiment 1.

Results:

The results are given as extinction at 450 nm and are summarized inTable 2.

TABLE 2 Day 0 24 38 52 66 Antigen n MW σ MW σ MW σ MW σ MW σ 0.9% (w/v)NaCl 10 0.015 0.002 0.019 0.004 0.019 0.006 0.014 0.002 0.024 0.005solution His-m-OspC_(VS461) 10 0.014 0.003 0.017 0.003 0.069 0.114 0.7220.986 2.195 1.518 His-m-Cys-OspC_(VS461) 10 0.014 0.002 0.036 0.0241.043 0.995 1.892 1.288 3.868 0.202

It is found that considerably faster and higher measurement values aregenerated if the animals have been immunized with the variantHis-m-Cys-OspC_(VS461). It is furthermore found that at the end of theimmunization programme, all the sera of animals immunized with thevariant His-m-Cys-OspC_(VS461) generate high measurement values close tothe mean. In contrast, very low or low measurement values in some of theanimals which were immunized with the variant His-m-OspC_(VS461)indicate that the immunization in these animals did not lead to asubstantial antibody titre.

The incubation on EUROLINE-WB strips shows that all the sera whichgenerate an increased extinction (E_(450 nm)≧0.3) in ELISA generate aband at the migration site of the OspC. In this context, the bandintensity approximately correlates with the extinction.

The two results taken together suggest that the mice which wereimmunized with the variant His-m-Cys-OspC_(VS461) generate antibodiesfaster than after immunization with the variant His-m-OspC_(VS461), andthat higher antibody concentrations are achieved at the end of theimmunization.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

LITERATURE LIST

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1. A vaccine against a Borrelia infection comprising a protein, saidprotein comprising a first OspC polypeptide and a second OSpCpolypeptide, wherein the first OspC polypeptide is linked to the secondOspC polypeptide via a disulphide bridge.
 2. The vaccine according toclaim 1, wherein the first OspC polypeptide, the second OspCpolypeptide, or both the first and second OspC polypetides have an aminoacid identity of at least 90% with SEQ ID NO:3, SEQ ID NO:6 or SEQ IDNO:9 and are specifically recognized by antibodies against OspC fromBorrelia.
 3. The vaccine according to claim 1, wherein the first OspCpolypeptide, the second OspC polypeptide, or both the first and secondOspC polypeptides comprise at least one epitope which is specificallyrecognized by an antibody against OspC from Borrelia , the epitopecomprising at least a partial sequence of 10 contiguous amino acids fromSEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9.
 4. The vaccine according toclaim 3, wherein the at least 10 contiguous amino acids from SEQ IDNO:3, SEQ ID NO:6 or SEQ ID NO:9 are the 10 C-terminal amino acids ofthese sequences and said at least 10 contiguous amino acids are in theC-terminal location of the OspC polypeptide.
 5. The vaccine according toclaim 1, wherein the first OspC polypeptide, the second OspCpolypeptide, or both the first and second OspC polypeptides do notcomprise any recognition sequence for an acylation.
 6. The vaccineaccording to claim 1, wherein the disulphide bridge is formed by linkingof a cysteine which is not more than 30 amino acid positions away fromthe N-terminus of the first OspC polypeptide, the second OspCpolypeptide, or both the first and second OspC polypeptides.
 7. Thevaccine according to claim 1, wherein the first OspC polypeptide, thesecond OspC polypeptide, or both the first and second polypeptdiescomprise a sequence according to SEQ ID NO:2, SEQ ID NO:5 or SEQ IDNO:8.
 8. The vaccine according to claim 1, wherein by means of thedisulphide bridge a homodimer of two identical OspC polypeptides or aheterodimer of different OspC polypeptides is formed.
 9. The vaccineaccording to claim 1, wherein the recognition of the polypeptide byantibodies against OspC from Borrelia is reduced by bringing it intocontact with at least one thiol-containing reagent, optionally incombination with an alkyl halide. 10.-16. (canceled)